Doctor of Philosophy / Plant Pathology / Frank F. White / Rice and wheat are two major crops that suffer losses from the diseases of bacterial blight and bacterial leaf streak, which are caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas translucens pv. undulosa (Xtu), respectively. Transcriptional-Activator Like (TAL) effectors, a special family of type III effector proteins from Xanthomonas, have been demonstrated as critical virulence factors that act by inducing corresponding susceptibility (S) genes in several disease complexes of plants. In this study, I analyzed the contributions of TAL effectors from Xoo and Xtu to virulence and in modulating host gene expression to enhance susceptibility. Specifically, the TalC effector from the African Xoo strain AXO1947 was identified as a critical virulence factor, which functions by promoting expression of the gene OsSWEET14 in rice. TalC is interchangeable with other major TAL effectors from Asian strains of Xoo on the basis of functional complementation. The TAL effector PthXo2 from the Asian Xoo strain JXO1 is a major virulence factor and contains 21.5 repeats in the central repetitive region that targets OsSWEET13 in indica rice varieties but not in japonica rice varieties. A one repeat deletion in the PthXo2 effector enabled effector specificity to switch from indica rice to japonica rice. TAL effector genes from a genomic analysis of the Xtu strain XT4699 and related strains were characterized with regards to their involvement in virulence and the modulation of host gene expression in the Chinese Spring wheat cultivar. The identification of TAL effectors with virulence contributions and their target S genes is important for understanding the virulence mechanisms of Xanthomonas bacteria and promises to provide new strategies for disease control.

Master of Science / Entomology / C. Michael Smith / The bird cherry oat aphid, Rhopalosiphum padi, and one of the viruses it vectors, barley yellow dwarf virus (BYDV), form a very damaging pest unit on cereals. Understanding how people can better manage crops to prevent damage or recognize environmental or geographic factors that put their crops at risk for BYDV could lead to improved virus aphid management strategies. One of the most successful methods for mitigating pest damage is using pest-resistant varieties of crops. Seven candidate wheat varieties were screened for resistance to R. padi, by testing aphid population densities and aphid host choice. Results of this research identified six varieties of wheat that show resistance to R. padi. One of the major knowledge gaps in BYDV management is forecasting potential damage. Our objective was to create viral presence maps, to start building the foundation of correlations between persistent and changing frequency of viruliferous R. padi. The results of BYDV assays in R. padi in wheat fields across Kansas indicated that the amount of BYDV infection in viruliferous aphids changes rapidly from year to year, and differs considerably between geographic regions and field landscape characteristics in Kansas. Neonicotinoid wheat seed treatment is a management technique that reduces R. padi populations. However, a common question among producers is whether or not seed treatments stop viral transmission. Results of greenhouse seed treatment experiments with plants from neonicotinoid treated and untreated seed infested with viruliferous R. padi indicated no significant difference in viral transmission due to seed treatment. This new information about R. padi wheat varietal resistance, geographic distribution of BYDV in Kansas, and neonicotinoid seed treatment creates a better understanding of aphid-virus-wheat interactions. Results from this thesis directly enhance producer ability to forecast risk from BYDV, select wheat varieties that effectively reduce R. padi as a BYDV vector, and to make better decisions about the use of insecticide to reduce BYDV infection.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Doctor of Philosophy / Department of Plant Pathology / Scot H. Hulbert / The rice cultivar Oryzica Llanos 5 (OL5) possesses a high level of resistance to the fungus Magnaporthe grisea. The number and chromosomal location of quantitative trait loci (QTL) conferring resistance against eight isolates of the blast fungus were tested in two different populations of recombinant inbred lines from the cross Fanny x OL5. Twenty one QTL were detected and associated with the resistance traits, disease leaf area and lesion type, on 9 rice chromosomes. Eight of these 21 resistance loci had significant resistance effects in both experiments, while the others had effects that were only statistically significant in one experiment. Most, but not all, of the QTL occurred in the same genomic regions as either genes with major race-specific effects or other resistance QTL that had been described in previous experiments. Most of the QTL appeared to be race-specific in their effects but it is possible some of the QTL with smaller effects were nonspecific. One of the blast isolates used was FL440, which causes limited disease on OL5 and was probably virulent on most or all of the major genes from OL5. Three QTL affected resistance to FL440 in both experiments, one of which mapped to a region on chromosome 9 where no blast resistance genes have yet been mapped. An advanced backcross strategy with marker-assisted selection for OL5 alleles in QTL regions was used to generate five BC2F3 populations carrying five different target regions associated with partial resistance to rice blast disease. Three of five of these populations were analyzed for segregation for resistance to the M. grisea isolate FL440. One QTL designated qrbr-11.3 near the bottom of rice chromosome 11 was found to be significantly associated with partial blast resistance in 120 lines of a BC2F3 population (P< 0.01). This QTL accounted for 12.4% and 8.0% of the phenotypic variation in diseased leaf area and lesion type observed under greenhouse inoculation. Examination of the genomic sequence at the qrbr-11.3 locus showed that twenty-nine candidate resistance genes are present at that locus (~1.8 Mb), twenty-seven of which are predicted NBS-LRR genes. Ultimately, the information from this study can be integrated into the development of improved lines with OL5-derived QTL for resistance.

Doctor of Philosophy / Department of Plant Pathology / Jianmin Zhou / Plants activate innate immune responses or innate immunity upon pathogen infection. There are two types of plant innate immunity: PAMP-triggered innate immunity (PTI) and effector-triggered innate immunity (ETI). The molecular basis for ETI has been well documented. However, the study on PTI and its interplay with pathogen virulence is in its infancy. My research focuses on the interplay between PTI and bacterial virulence in Pseudomonas-Arabidopsis interactions. NHO1, a gene required for nonhost resistance to Pseudomonas syringae, encodes for the 3-glycerol kinase in Arabidopsis genome. NHO1 functions, at least in part, by depriving glycerol from nonhost bacteria cells. NHO1 is induced by a well-known bacteria PAMP flg22. The induction of NHO1 correlates well with the resistance against Pseudomonas syringae pv. tabaci because a mutant strain of P. s. pv. tabaci deficient in NHO1 induction gains partial virulence on Arabidopsis plants. P. s. pv. tomato strain DC3000 induces transient NHO1 expression that is suppressed in a type III secrection system-dependent manner. Using protoplast assay, nine DC3000 effectors that are able to suppress NHO1 were identified. One of them, HopAI1, induces leaf chlorosis and helps nonpathogenic bacterial growth when expressed in Arabidopsis plants, suggesting that HopAI1 has virulence activity in planta. To study AvrB virulence activity in Arabidopsis plants, one mutant compromised in AvrB-specific RAR2.6 induction has been characterized in detail. rrb3 is more susceptible to a nonhost bacteria P. s. pv. tabaci strain 6505, a virulent bacteria P. s. pv. tomato strain DC3000 and an avirulent bacteria strain DC3000 (avrB). The mutant allele rrb3 carries a point mutation at the end of RAR1 CHORD II domain. RRB3 (RAR1), together with NDR1, is involved in the type II nonhost resistance to P. s. pv. tabaci but not in the type I nonhost resistance to P. s. pv. phaseolicola. RAR1 participates in basal resistance against DC3000 by antagonizing COI1 activity. AvrB targets RAR1 to trigger AvrB-dependent leaf chlorosis and enhanced bacterial growth. The AvrB-dependent enhanced bacterial growth but not leaf chlorosis requires COI1, suggesting that AvrB targets JA signaling pathway to promote parasitism.

Master of Science / Department of Plant Pathology / Karen A. Garrett / While plants are typically exposed to multiple stressors in the field, studies of genome-wide gene expression and phytohormone responses in wild plant species exposed to multiple stressors are rare. Our objectives were to determine the effects of drought and rust stress on gene expression in Andropogon gerardii, the dominant grass in tallgrass prairie, and associated levels of phytohormone production. In a factorial design, plants experiencing drought or non-drought conditions were either inoculated with the rust pathogen Puccinia andropogonis or not inoculated. Gene expression was evaluated with maize microarrays. Drought-stressed plants significantly decreased expression of genes associated with photosynthesis and the hypersensitive response, while expression of genes associated with chaperones and heat-shock proteins increased. No significant differences in gene expression in response to the rust treatment were detected using a mixed model analysis of variance and false discovery rate protection, probably because of the low infection rate. Phytohormone production increased when both stresses were present. The rust treatment significantly increased benzoic acid (BA) production in the presence of drought, while the drought treatment alone significantly increased salicylic acid (SA) production. Leaf tips usually had higher levels of all phytohormones in all treatments and the leaf section evaluated had a larger effect on phytohormone level than did the treatments applied.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Edward E. Carey / With acceptance and utilization of chemical pesticides declining, some vegetable producers are turning to alternative methods to manage plant health issues. Compost tea (CT) has provided control of some foliar pathogens and may provide benefits beyond disease suppression. Despite an increasing body of popular and scientific literature focusing on CT as a biological control option for growers, information on the efficacy of CT is still lacking for many pathosystems. In this study, field trials were conducted to evaluate the efficacy of CT on Septoria lycopersici, causal agent of Septoria leaf spot on tomato, in Kansas, in 2006 and 2007. Previous research done at KSU with a similar CT showed adequate control of this pathogen in field and greenhouse studies conducted. Additional work to develop a rapid screening method for efficacy of CT formulations was carried out in the greenhouse at Manhattan, KS. CT sprayed weekly on tomato plants prior to and after disease onset led to no significant difference in control of the pathogen compared to untreated controls. A contact fungicide (chlorothalonil) provided significant control of the pathogen in 2007, but not in 2006. These results contrast with those obtained in previous K-State research. It is difficult to assess why such striking differences were obtained, but the variation in these results point to the need to identify optimal recipes of CT for this pathosystem. Preliminary investigations standardized plant age, inoculum concentration, incubation conditions, and incubation interval for measurable Septoria leaf spot disease development on young tomato plants in the greenhouse. Ingredients of the field-tested CT were used to make a variety of CTs to test using the greenhouse-screening assay. Further work on identifying effective CT recipes is needed to substantiate the validity of this screening protocol and to evaluate the correlation of this method with disease suppression in the field.

Doctor of Philosophy / Department of Plant Pathology / Robert L. Bowden / John F. Leslie / Gibberella zeae is an ascomycete filamentous fungus and the major cause of Fusarium head blight, also called scab, in small grains. This dissertation contains three related studies of G. zeae. In the first, the genetic map was aligned with the first assembly of the genomic sequence released by The Broad Institute (Cambridge, MA). Approximately 99% of the sequence was anchored to the genetic map, indicating the high quality of the sequence assembly and validity of the genetic map. The alignments grouped the linkage groups and supercontigs into four sets, which is consistent with the hypothesis that there are four chromosomes in this fungus. In the second, the sex pheromone precursor genes (ppg1 and ppg2) and the pheromone receptor genes (pre1 and pre2) were identified and characterized. Deletion of ppg1 or pre2 ([Delta]ppg1 or [Delta]pre2 strains) reduced the number of perithecia produced by self-fertilization, but did not completely block perithecial formation. The proportion of crosses resulting from outcrossing increased when the [Delta]ppg1 strains were used as the female in crosses with male strains containing an intact ppg1 gene. [Delta]ppg2 and [Delta]pre1 mutants had no discernable effect on morphological phenotype or self-fertilization. Thus, one of the pheromone/receptor pairs (ppg1/pre2) found in many Ascomycetes has a role in, but is not essential for, selfing or outcrossing in G. zeae, whereas the other pheromone/receptor pair (ppg2/pre1) no longer has a detectable function in sexual reproduction. In the third study, spore germination of G. zeae was tested in the presence of α- factor-like pheromone peptides of G. zeae or N. crassa. The pheromone peptide of N. crassa more efficiently inhibited spore germination than did the peptide from G. zeae. Arginine and lysine residues were the most important determinants in blocking spore germination. In conclusion, this research has validated the genetic map and the genomic assembly of G. zeae, characterized sex pheromone functions and characterized pheromone peptide ability to inhibit spore germination. The pheromone peptides of G. zeae and N. crassa may be useful as control agents for G. zeae and pheromone peptide efficacy might be further enhanced by judicious substitutions for some of the amino acids.

Doctor of Philosophy / Department of Plant Pathology / William W. Bockus / James P. Stack / Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae (MoT), is a serious disease of wheat causing yield failures and significant economic losses during epidemic years in Brazil, Paraguay, and Bolivia. Although outbreaks occur only sporadically, wheat blast is considered a major disease affecting wheat production in South America and may be a threat to the wheat crop in the United States. Wheat is a major crop in the U.S. and wheat exports from the U.S. are important to food security of several countries around the World. Thus, it is important to understand the potential for MoT entry and establishment into the U.S. and to test U.S. wheat cultivars for susceptibility to MoT. The hypotheses of this research project were a) importing wheat grain from Brazil does not pose a risk for MoT establishment in the U.S., and b) resistance to MoT head infection does not exist in U.S. hard red winter wheat elite cultivars. Quantitative pathway analysis models were used to estimate the risk of MoT entry and establishment, in the coterminous U.S. and in a more targeted area within southeast North Carolina, via the importation of wheat grain from Brazil. The pathway model predicted that significant risk for MoT entry and establishment exists in some areas of the U.S. However, in approximately 60% of the coterminous U.S. winter wheat production areas the risk of MoT establishment was estimated to be zero. With respect to winter wheat growing areas in the U.S., conditions for MoT establishment and wheat blast outbreak occur only in small, restricted geographic areas. A higher resolution pathway analysis based on a ground transportation corridor in North Carolina indicated that conditions for MoT establishment exist seven out of ten years. Among U.S. cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars PostRock, Jackpot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection.

Doctor of Philosophy / Department of Plant Pathology / John F. Leslie / Members of the genus Fusarium are widely distributed in many geographic regions of the world. This genus includes plant pathogens of many important cereal crops, e.g., wheat, maize, rice and sorghum, and of other native and economically important plants. From culture collections at Kansas State University and Universiti Sains Malaysia, strains from Southeast Asia, primarily from Malaysia and Thailand, associated with mango malformation disease, bakanae disease of rice, and stalk rot of sorghum were analyzed in sexual crosses and molecular diagnostics, e.g., Amplified Fragment Length Polymorphisms (AFLPs). Fusarium proliferatum was recovered from all three crops, with each crop also yielding some species unique to the crop, e.g. F. fujikuroi from rice, F. thapsinum from sorghum, and F. mangiferae from mango. These results are consistent with hypotheses that F. proliferatum has a wide host range while other species have much more limited host preferences. The absence from our samples of species associated with these diseases in other parts of the world suggests policies should be developed to reduce the chances of introduction of novel pathogens into Southeast Asia. Fusarium fujikuroi and F. proliferatum are closely related. They usually can be separated by sexual cross-fertility and DNA sequence analysis. However, some strains can cross irregularly and with poor fertility to produce viable interspecific hybrids. From a laboratory cross between F. fujikuroi FGSC8932 and F. proliferatum FGSC7615, 533 progeny were collected. These progeny were characterized for their AFLP genotype, mating type, gibberellic acid production, and pathogenicity on rice, onions, and apples. A recombination-based map from this interspecific cross was constructed. QTLs associated with gibberellic acid production, rice pathogenicity, and onion pathogenicity were identified. Gene segregation amongst the progeny of the F. fujikuroi × F. proliferatum cross was distorted towards F. proliferatum. Both novel and transgressive pathogenicity phenotypes were detected. Overall, this research demonstrates the potential threats that can result from an interspecific cross. These threats include pathogens with novel toxin profiles, new pathogenicity phenotypes, and more virulent strains. The variation observed among the progeny may enable isolation and characterization of genetic factors that have a role in pathogenicity, toxin production, and host specificity.